Effects of Heparin on Topical Use of Plasters Containing a Non-Steroidal Anti-Inflammatory Drug

ABSTRACT

The present invention provides for a method of improving the release of non-steroidal anti-inflammatory drugs (NSAIDs) from a plaster or bandage comprising an adhesive layer with a pharmaceutically acceptable NSAID together with heparin or a heparinoid. The invention also provides for methods of reducing muscle hyperalgesia in subjects without spontaneous pain.

FIELD OF INVENTION

Plasters for topical use having an analgesic activity and being able toinduce the re-absorption of hematomas, containing a non-steroidalanti-inflammatory drug in association with heparin or a heparinoid.

PRIOR ART

U.S. Pat. No. 6,592,891 and U.S. Pat. No. 7,799,338, incorporated hereinby reference in their entireties, describe plasters for topical usehaving an analgesic activity and at the same time being capable toinduce reabsorption of hematomas.

These plasters comprise a substrate layer, an adhesive layer in the formof a hydrogel matrix containing a pharmaceutically acceptablenon-steroidal anti-inflammatory drug (“NSAID”), heparin or a heparinoidand a protective film which can be removed at the moment of use.

Applicants have now surprisingly found that plasters containing NSAIDsmixed with heparin or a heparinoid are more effective in increasing painthresholds to electrical stimulation of cutis, sub cutis and muscle inasymptomatic subjects with a latent algogenic condition compared toplasters containing NSAIDs only.

Moreover, Applicants have also surprisingly found that heparin or aheparinoid, without being released by the plaster in significant amount,is capable of increasing the release of the NSAIDs from the plaster andits permeation through a synthetic membrane.

DESCRIPTION OF THE INVENTION

It has been found that plasters comprising heparin and a NSAID have abetter effect on pain thresholds to electrical stimulation of cutis, subcutis and muscle when topically applied in subjects without spontaneouspain compared to plasters comprising NSAID only.

Moreover, it has also been found that heparin increases the permeabilityand the release of NSAID present on an adhesive layer of a plasterwithout being released by the plasters.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the permeation profiles of diclofenac from Flector® Patchand Flector-Heparin Patch.

DETAILED DESCRIPTION OF THE INVENTION

It has surprisingly been found that the use of heparin or a heparinoidon a plaster or bandage together with a NSAID improves the release ofthe NSAID from the plaster. It has also surprisingly been found that thepresence of heparin or a heparinoid on a plaster or bandage increasesthe membrane permeability of the NSAID.

Moreover, unexpectedly, Applicants have also found that topicalapplications of a plaster or bandage containing heparin, or heparinoidtogether with a NSAID are more effective in reducing muscle hyperalgesiain subjects without spontaneous pain (as evidenced and quantified by theincrease in pain threshold to external standardized electrical orpressure stimuli) than topical applications of plasters containing onlya NSAID.

Accordingly, the present invention provides for methods of improvingrelease of non-steroidal anti-inflammatory drugs (NSAIDs) from a bandagecomprising:

a) a substrate layer;

b) an adhesive layer in the form of a polymeric hydrogel matrixcomprising an effective amount of a pharmaceutically acceptable NSAIDand heparin or a heparinoid, at least one thickening agent, at least onewetting agent, at least one cross-linking agent; and

c) a protective film which can be removed at the moment of use, whereinsaid hydrogel matrix further comprises a surfactant consisting of sodiumethylenediamine tetraacetate; said method comprising

topically applying said bandage to patients in need thereof; and

assessing an improved NSAID release compared NSAID release from bandageswithout heparin.

The present invention provides also for methods of increasing membranepermeation of non-steroidal anti-inflammatory drugs (NSAID) from abandage comprising:

a) a substrate layer;

b) an adhesive layer in the form of a polymeric hydrogel matrixcomprising an effective amount of a pharmaceutically acceptable NSAIDand heparin or a heparinoid, at least one thickening agent, at least onewetting agent, at least one cross-linking agent; and

c) a protective film which can be removed at the moment of use, whereinsaid hydrogel matrix further comprises a surfactant consisting of sodiumethylenediamine tetraacetate; said method comprising:

applying said bandage to a membrane; and

assessing an increased membrane permeation of NSAID compared to membranepermeation obtainable from bandages without heparin.

It provides also for methods of reducing muscle hyperalgesia in subjectswithout spontaneous pain, said method comprising:

assessing an initial pain threshold in said subjects;

applying to said subjects a bandage comprising:

a) a substrate layer;

b) an adhesive layer in the form of a polymeric hydrogel matrixcomprising a pharmaceutically acceptable NSAID and heparin or aheparinoid, at least one thickening agent, at least one wetting agent,at least one cross-linking agent; and

c) a protective film which can be removed at the moment of use, whereinsaid hydrogel matrix further comprises a surfactant consisting of sodiumethylenediamine tetraacetate;

reassessing the pain threshold; and

determining a reduced muscle hyperalgesia in said subjects measured as adifference between the initial and the reassessed pain threshold.

According to one embodiment of the present invention, the NSAID isselected from the group consisting of diclofenac, diflunisal, ibuprofen,ketoprofen, naproxen, acetysalicic acid, salsalate, celecoxib, etodolac,fenoprofen, flurbiprofen, indomethacin, ketorolac, meloxicam, mefenamicacid, nabumetone, oxaprozin, piroxicam, rofecoxib, sulindac, tolmetin,valdecoxib or pharmaceutically acceptable salts thereof.

According to a particularly preferred embodiment the NSAID is diclofenacin the form of a pharmaceutically acceptable salt, and more preferably,it is a salt with a heterocyclic amine of general formula

where m is 0 or 1.

According to a better embodiment, the heterocyclic amine isN-hydroxyethyl pyrrolidine (epolamine).

The diclofenac salt is contained in the plaster according to the presentinvention in concentrations generally ranging from 0.1 to 5 wt %,preferably in concentrations of between 0.3 and 3 wt % with respect tothe total weight of the composition used for the preparation of thehydrogel matrix.

According to a particularly preferred embodiment, the concentration ofthe diclofenac salt is 1.3 wt % with respect to the total weight of thecomposition used for the preparation of the hydrogel matrix.

When the plaster according to the present invention contains aheparinoid, the latter preferably presents a molecular weight of between5,000 and 30,000 DA.

The heparin or heparinoid is present in concentrations such that itstotal quantity in the plaster is between 0.05 and 1%, which correspondsrespectively to a concentration range of between 1,400 and 28,000IU/plaster (100-2,000 IU/g matrix). According to a particularlypreferred embodiment, heparin is contained in concentrations such thatthe corresponding content is 5600 IU per plaster.

It is advisable that the composition used for preparing the hydrogelmatrix should present pH values of between 7.2 and 9, preferably ofbetween 7.5 and 8.5. At pH values lower than 7.2, the diclofenaccrystals that are insoluble in water precipitate; values higher than 9may cause irritation of the skin. To adjust the pH of the hydrogelcomposition, any organic or inorganic acid, or any organic or inorganicbase may be used, without any particular limitation. The concentrationof the above-mentioned acid or base is not critical either and may varyaccording to the pH value that the hydrogel composition reaches.

In addition to the aforementioned active principles, the hydrogel matrixfurther contains thickening agents, wetting agents, fillers,preservatives, cross-linking agents, surfactants, stabilizers, and thelike.

Preferably, the composition used for the preparation of the hydrogelmatrix contains as thickening agents the following: polyacrylic acid,sodium polyacrylate, sodium carboxymethyl cellulose, polyvinyl alcohol,polyvinyl pyrrolidone, gelatine or corresponding mixtures. Theconcentration of the above additives is generally between 3 and 30 wt %,preferably between 5 and 20 wt %. If the concentration is lower than 3wt %, the viscosity of the composition is too low, so that thecomposition comes out of the plaster and remains on the skin once theplaster is applied; on the other hand, if the concentration is too high,it is not very workable. According to a particularly preferredembodiment, the thickening agents are a mixture consisting of thefollowing: gelatine, polyvinyl pyrrolidone, sodium polyacrylate, andsodium carboxymethyl cellulose in a total concentration of 9 wt % withrespect to the total weight of the hydrogel matrix. The hydrogel matrixof the plaster according to the present invention preferably comprisesat least one wetting agent chosen from among glycerol, propylene glycol,polyethylene glycol, 1,3-butanediol, and an aqueous solution ofD-sorbitol, preferably in a concentration of 70 wt %.

The concentration of the said wetting agents in the composition used forthe preparation of the hydrogel matrix according to the presentinvention is between 5 and 70 wt %, preferably between 10 and 60 wt %with respect to the total weight of the composition used for thepreparation of the hydrogel matrix.

If the quantity of wetting agent is lower than 5%, the wetting effect isnot sufficient and the composition dries quickly; if, instead, thequantity of wetting agent is higher than 70%, mixing of the componentsis difficult.

The cross-linking agent is preferably an aluminum or calcium compoundpresent in the composition used for the preparation of the hydrogelmatrix in a concentration of between 0.01 and 3.0 wt %. If the quantityof this additive is lower than 0.01 wt %, cross-linking is insufficient,so that the resistance to heat of the hydrogel matrix is reduced;consequently, two drawbacks may occur, either during storage or duringapplication: during storage, the composition is too fluid and comes outof the plaster in the sterile container in the sterile container of thelatter; when the plaster is applied, the composition leaves a residue onthe skin. When the concentration of the cross-linking agent is higherthan 3%, the rate of cross-linking is too high and consequently theviscosity of the composition used for the preparation of hydrogel matrixincreases, so that the corresponding workability decreases. As a filler,one of the following, for instance, may be used: kaolin, titaniumdioxide, bentonite, or mixtures of the said compounds.

As a preservative, the hydrogel matrix that is the subject of thepresent invention may contain either preservatives of a conventionaltype, such as the esters of para-alkyloxy benzoic acid, for exampleNipagin and Nipasol, or sorbic acid. The hydrogel matrix may possiblycontain a surfactant, such as a polyoxyethylene sorbitan ester (Tween80) and a stabilizer, such as sodium ethylenediamine tetraacetate. Asfar as the substrate layer is concerned, any material usually employedfor this purpose may be used, such as fabric, non-woven fabric, paper,plastic film and corresponding laminates. As regards the removableprotective film, this may be of a conventional type, for instance,siliconized paper, or may be made of a plastic material, such aspolyethylene, polyethylene terephthalate, or polyvinyl chloride.

The present plaster is prepared according to conventional methods which,in particular, envisage the following fundamental stages: mixing of thevarious components of the composition used for the preparation of thehydrogel matrix, co-extrusion of the hydrogel matrix between thesubstrate layer and the removable protective film.

The mixing phase is in particular conducted in the following stages:1-A) mixing of one part of water with the filler and with part both ofthe cross-linking agent and of the thickening agent; 1-B) subsequentaddition to the mixture obtained in stage (1-A) of further thickeningagents; 1-C) addition to the mixture obtained in stage (1-B) ofpreservatives, a stabilizer, a pH adjuster, as well as of the remainingpart of the cross-linking agent and thickening and wetting agents; 1-D)addition to the mixture obtained in stage (1-C) of the pharmaceuticallyacceptable diclofenac salt; 1-E) addition to the mixture obtained instage (1-D) of the heparin or heparinoid.

The preparation of the hydrogel matrix used as adhesive layer of theplaster according to the present invention is described in U.S. Pat. No.6,592,891 and in U.S. Pat. No. 7,799,338 incorporated herein byreference in their entireties.

The following examples exemplify the methods described herein. Theexamples are for purposes of illustration only and are not intended tobe limiting of the invention.

Example 1 Effect of Heparin on the In Vitro Permeability of DiclofenacEpolamine

Permeation of diclofenac epolamine (DHEP) was assessed in vitro withFlector® and Flector-Heparin patches by using a Franz Cell System.

According to the non parametric method of the FDA Guidance, theFlector-Heparin Patch is not equivalent to Flector® Patch in terms ofDHEP release and permeability. Mean DHEP permeation flow rate over the24 hours was higher from Flector-Heparin Patch than from Flector® Patch(2.01±0.60 vs. 0.86±0.19 μg/cm²/h).

Also, the cumulative amount of DHEP that crossed the Franz Cell siliconemembrane over 24 hours was significantly greater from Flector-HeparinPatch than from Flector® Patch (48.23±14.37 vs. 20.54±4.68 μg/cm²).Heparin did not appear to be released from the plaster.

In conclusion, the results of the in vitro permeation tests showed thatheparin contained in Flector-Heparin Patch enhances DHEP permeation andsuggest that an improved local anti-inflammatory and analgesic activitymay be expected upon Fleeter-Heparin Patch application to the skin.

Applicants have developed two medicated patches containing diclofenacepolamine (DHEP) as an active ingredient, namely Flector Patch andFlector-Heparin Patch.

The two patches have identical qualitative and quantitative composition,except for the content of heparin sodium (40,000 IU/100 g of paste or5,600 IU/plaster), a natural glycosaminoglycan obtained by extraction ofporcine intestinal mucosa, which is present only in the Flector-HeparinPatch. Both patches consist of 14 g of paste, containing 181 mg of DHEP,applied to an unwoven cloth of 10 cm×14 cm in size (surface area 140cm²).

According to the FDA guidance for Industry on Non sterile SemisolidDosage Forms—CDER, May 1997′, the evaluation of in vitro release of DHEPfrom topical dosage forms can be performed through the use of an openchamber diffusion cell system, i.e., a Franz Cell System, fitted with asynthetic membrane. This system was used to compare DHEP release andpermeability from the two patches in an attempt to explain the role ofheparin in the enhancement of clinical activity observed in severalclinical trials performed to date. The release and permeability ofheparin was also investigated.

The comparison of DHEP release from the two patches and membranepermeability was expected to demonstrate whether heparin, which isunlikely to be either released from the patch or permeate the skinbarrier due to its high polarity, negative charge and molecular weight,may act as an enhancer of DHEP release from the patch, thus providingthe medicated plaster with higher anti-inflammatory and analgesicactivity when locally applied.

The study was conducted using a Franz Cell Apparatus equipped with asynthetic membrane. Concentrations of DHEP and heparin were determinedby a validated HPLC method and a biological assay, respectively.

Equipment:

Permeation equipment: Franz Cell System. Standard, open cap, flat groundglass surface with 15 mm diameter orifice, total diameter of cell 25 mm,area 1.767 cm². Microette Plus—Hanson; Multifill Hanson—FractionCollector System

Silicone membranes: Perouse Plastie, PlastiePerthese LP 800-5, 0.5 mmthickness, of appropriate size (25 mm diameter) to fit the diffusioncell. Lipophilic nonporous silicone membranes may be useful surrogatemembranes for human skin when evaluating in vitro releasecharacteristics of a drug from topical or transdermal patchformulations.

HPLC: Agilent 1200; software EXChrom Elite.

pH meter: Metrohm 827 pH lab.

Analytical Balance: Mettler XP105DR.

Spectrophotometer UV-Vis: Jasco V-630.

Vortex: IKA MS3.

Thermostatic Bath: Julabo basic.

For the assessment of DHEP permeability, the Reference patch (R,Flector®) and the Test patch (T, Flector-Heparin) were taken from theiroriginal envelopes (primary packaging) immediately before the study, cutinto pieces with shape (circular) and size (25 mm diameter)corresponding to those of the synthetic membrane in order to properlyfit them into the Franz cells.

The amount of DHEP in the Flector® Patch and the Flector-Heparin Patchis 181 mg, the total patch area is 140 cm², and the amount of DHEP persurface unit is 1.293 mg/cm² (corresponding to 1 mg/cm² diclofenacsodium salt). Considering that the surface area of a donor Franz Cellorifice is 1. 767 cm² (cell permeation area), the amount of DHEP exposedto the silicone membrane per cell was 2.284 mg.

Samples of R and T were placed on the upper side of the membrane in theopen donor chamber of the diffusion cell and a receptor medium (PBS pH7.4, total volume 7 mL) was placed on the other side of the membrane inthe receptor cell. Diffusion of DHEP from the patch samples across themembrane was monitored by assay of sequentially collected samples ofreceptor fluid. The removed aliquots of the receptor phase were replacedwith fresh aliquots of the buffer. Each collected aliquot (total volume2.5 ml, sample volume 1.0 mL) was immediately analyzed for DHEP contentby HPLC. The amount of DHEP released from each sample of Flector w/woHeparin was calculated by the following equation, which takes intoaccount the amount of DHEP subtracted from the receiving buffer withsample withdrawals.

$Q_{n} = \frac{\lbrack {( {C_{n}*7} ) + {\sum\limits_{x = 1}^{x = {n - 1}}( {C_{x}*V_{p}} )}} \rbrack}{1.767}$

Where:

Q_(n)=amount of drug permeated at time n (μg/cm²);

Cn=DHEP concentration in sample taken at time n (μg/mL);

n=sampling times, i.e., 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 20, 24 hours;

7=total cell volume (mL);

C_(x)=DHEP concentration at time x (μg/mL), where x corresponds, atsampling time n, to sampling time n−1;

Vp=withdrawal volume (2.5 mL)=rinsing volume (1.5 mL)+sampling volume(1.0 mL);

1.767 (cm²)=cell permeation area (15 mm diameter).

In order to validate the synthetic membrane for the permeation ofheparin, a preliminary experiment was conducted by placing a heparinsolution in the 5 donor chambers (T) and determining the passage throughthe silicone membrane into the receiving chamber. Heparin solution inthe donor chamber was constituted by 1 mL of a 70 IU/mL sodium heparinsolution in PBS pH 7.4. The heparin solution concentration wasconsistent with the amount of heparin exposed to the silicone membranewhen patch samples were tested. A blank sample (B, 1 mL of PBS pH 7.4,without heparin) was also placed in a donor chamber.

Heparin concentration was determined by a method recommended by USP 30,Official monographs, page 2274 (Heparin sodium-Anti-factor Xa,activity), using a Stachrom Heparin Kit (Diagnostica SIBSA TAGO). Themethod was adapted for application to Franz Cell permeated samples andvalidated. Briefly, heparin, in the presence of AT III (Antitrombin III)excess binds ATIII to form a complex. By adding the coagulation factorXa, a three element complex is formed, together with the release ofresidual factor Xa. This residual factor hydrolyzes a chromogenicsubstrate, thus releasing para-nitroaniline (pNa) which is quantified byspectrophotometric analysis at λ=405 nm. The absorbance measured isinversely proportional to the heparin content in the sample. Maximumabsorption is detected in the sample without heparin.

Heparin quantification was performed by constructing a 5-point linearcalibration curve with a concentration range of 0.10-0.50 IU/mL. At thebeginning of analytical session, a calibration curve was freshlygenerated.

The analytical method showed a Limit of Quantitation (LOQ) of 0.10 IU/mLand a linearity concentration range of 0.10 IU/mL—0.50 IU/mL. Samplesolutions were analyzed undiluted or diluted with water to fulfilcalibration curve range. The results are reported in Table 1

Table 1 summarizes the results of the experiment in which a solution ofheparin was placed in the donor chamber of the Franz Cell System.

TABLE 1 Conc. Amount permeated Sample (IU/mL) (IU/cm²) Sample 1 (Blank)<LOQ ND Sample 2 <LOQ ND Sample 3 <LOQ ND Sample 4 0.108 0.43 Sample 50.141 0.56 Sample 6 0.177 070 ND = non-detectable

In 3 out of 5 test cells, heparin was detectable in the receivingchamber at concentrations close to LOQ (0.1 IU/mL). The results indicatethat this model is suitable to measure possible heparin permeability.Under the experimental conditions employed, heparin in solution showsnegligible permeability, if any.

Table 2 below reports the results of the experiment in which samples ofFlector-Heparin Patch were tested for heparin permeability in the FranzCell System. In this experiment, heparin was not detectable in thereceiving chamber. The combination of the two findings, i.e.,permeability of heparin from a solution (although negligible) but notfrom the patch, leads to the conclusion that heparin is not releasedfrom the patch.

TABLE 2 Conc. Amount permeated Sample (IU/mL) (IU/cm²) Sample 1 (Blank:Flector ®) <LOQ ND Sample 2 (Flector-Heparin) <LOQ ND Sample 3(Flector-Heparin) <LOQ ND Sample 4 (Flector-Heparin) <LOQ ND Sample 5(Flector-Heparin) <LOQ ND Sample 6 (Flector-Heparin) <LOQ ND ND =non-detectable

Tables 3 and 4 report the linear regression slopes that describe theDHEP permeation rate (μg/cm²/h), obtained from RUN 1 and RUN 2,resulting from the comparison test between Flector-Heparin Patch (Test,T) and Flector® Patch (Reference, R).

TABLE 3 Regression Slopes from RUN 1 SLOPE SAMPLE (μg/cm²/h) 1T 18.66162R 6.2625 3T 17.7630 4R 5.7784 5T 17.3546 6R 6.4551

TABLE 4 Regression Slopes from RUN 2 SLOPE SAMPLE (μg/cm²/h) 1R 6.17402T 15.4304 3R 5.3828 4T 13.1571 5R 6.7123 6T 14.0885

In this study, the permeation of diclofenac epolamine (DHEP) wasassessed in vitro with Flector® and Flector-Heparin patches by using aFranz Cell System, according to the FDA Guidance for Industry on Nonsterile Semisolid Dosage Forms⁽¹⁾. According to the non-parametricmethod of the FDA Guidance, Fleeter-Heparin Patch was not found to beequivalent to Flector® Patch in terms of DHEP release and permeability.The mean DHEP permeation flow rate over 24 hours was higher from theFlector-Heparin Patch than the Flector® Patch (2.01±0.60 vs. 0.86±0.19μg/cm²/h). Also, the cumulative amount of DHEP that crossed the FranzCell silicone membrane over 24 hours was significantly greater forFlector-Heparin Patch than Flector® Patch (48.23±14.37 vs. 20.54±4.68μg/cm²). Heparin did not appear to be released from the plaster.

In conclusion, the results of the in vitro permeation tests demonstratethat heparin contained in the FlectorHeparin Patch enhances DHEP releaseand subsequent membrane permeation. This observation is consistent withfindings from several phase 3 clinical trials of greater pain relief inminor soft tissue injury patients following Flector-Heparin Patchapplication to the site of injury, as opposed to treatment with theFlector® Patch.

Example 2

The experiment described in Example 1 was repeated using a Franz CellSystem fitted with a thinner silicone membrane (0.25 mm instead of 0.5mm) to allow greater passage of heparin from a control solution, withheparin content being determined as described in Example 1.

Table 5 below summarizes the results of the experiment in which aheparin solution was placed in the donor chamber of the Franz CellSystem.

TABLE 5 Conc. Amount permeated Sample (IU/ml) (IU/cm²) Sample 2 (cell 2)0.1107 1.11 Sample 3 (cell 3) 0.1247 1.25 Sample 4 (cell 4) 0.1517 1.52Sample 4 (cell 4) 0.1119 1.12 Sample 5 (cell 5) 0.1361 1.36 Conc. Blank(buffer pH 7.4) IU/ml % Heparin Permeated Sample 1 (cell 1) <LOQ ND ND =non-detectable

In all 5 tests cells (2 through 6), heparin was detected in thereceiving chamber at concentrations slightly above the LOQ (0.1 IU/ml).

In Table 6 below are reported the results of the experiment in whichsamples from Flector-Heparin Patch were tested to investigate heparinpermeability in the Franz Cell System. In this experiment, heparin wasnot detected in the receiving chamber (<LOQ).

TABLE 6 Conc. Amount of Heparin Permeated permeated Sample (IU/ml)(IU/cm²) Sample 1 (Blank: Fector ®) cell 1 <LOQ ND Sample 2(Flector-Heparin) cell 2 <LOQ ND Sample 3 (Flector-Heparin) cell 3 <LOQND Sample 4 (Flector-Heparin) cell 4 <LOQ ND Sample 5 (Flector-Heparin)cell 5 <LOQ ND Sample 6 (Flector-Heparin) cell 6 <LOQ ND ND =non-detectable

The results of this permeation study confirm the findings described inExample 1, in that all 5 patch receiving cells had a heparinconcentration below the limit of quantitation, while all those from theaqueous solution were above 1%, supporting the conclusion that, in theworst case, no more than 0.7 IUs of heparin could have left the patch.Furthermore, when residual heparin content was measured inFlector-Heparin Patch specimens following 24 hours of application to thesilicone membrane in five cells, the results indicated that the entireamount was retained (results not shown).

Example 3 Assessment of Residual DHEP and Heparin Content inFlector-Heparin Patches after Application to Healthy Subjects

a) DHEP Analysis

One Flector-Heparin patch was cut into pieces of 1 cm² and extractedwith methanol under reflux for one hour at 75° C. This procedure wasrepeated 3 times. The HPLC/UV analysis was performed with a Symmetryshield RP 18, 5 μm, 150×3.9 mm chromatography column and pH 3.5 acetatebuffer/acetonitrile (525/475 v/v) as mobile phase. The flow rate was 1.0mL/min and the UV detector wavelength at A 275 nm. The injection volumewas 20 μL and the retention time of diclofenac peak of 9.0 minutes±10%with area accuracy of <2% as from validation data.

The validation range of this method falls within the 80-120(%) of thedeclared DHEP content of patches. On the basis of the in vitropermeation study of DHEP, performed with a synthetic membrane, theexpected amount of DHEP absorbed in the skin application study would notbe higher than 20% of the declared content of the patch. Therefore,linearity, accuracy and precision of the DHEP assay analytical methodwere respected and this method was applied without generating amulti-point calibration curve.

b) Heparin Analysis

One Flector-Heparin patch was cut into pieces of 1 cm² and extractedwith 500 mL of Acetate buffer pH 7 under stirrer for 16 hours. Thesample preparation was performed by transferring 1 mL of suspension in aplastic vial and then it was centrifuged at 13000 rpm for 10 minutes.Heparin content was determined with HPLC-UV after enzymatic reactionwith Heparinase I at 37° C. for 3 hours.

Heparinase is selective for the cleavage of the glycosidic bond betweenhexosamines and uronic acids generating a double bond on thenon-reducing end of the uronic acid that absorbs at 232 nm permittingthe detection of oligosaccharides and disaccharides chains. After thereaction, the analysis was performed using a HPLC/UV system equippedwith a Siligel SAX 5 μm, 150×4.6 mm chromatography column withpre-column Uptistrategy C18-2, 5 μm, 10×4 mm/AGK 10/04-2. The mobilephase was sodium chloride 60 mM at pH 4.0 with sodium chloride 1.2 M atpH 4.0 combined in a gradient. The flow was 1 mL/min with UV detectorwavelength at λ. 232 nm and the injection volume 20 μL with areaaccuracy of <10% as from validation data. A validated analytical methodfor the heparin assay into Flector-Heparin patches was applied in thisstudy for the heparin residual dose measurements.

c) Results

Results are referred to patches applied to volunteers arms for 24 hours(Test) compared to patches of the same envelope not applied(references).

In details, one envelope, containing 5 patches, was provided for eachsubject. Two of 5 patches were applied by the investigator to the innerpart of the upper arms of the subject. One applied patch was analyzed toevaluate DHEP residual content, the other was analyzed for Heparinresidual content. One patch was analyzed at time zero of DHEP contentand one patch of the three remaining patches was analyzed as time zeroof Heparin content.

Table 7 shows the DHEP content value for 24 Flector-Heparin patchesanalyzed before topical treatment and after 24 hours topical applicationin 24 volunteers.

TABLE 7 Time 0 After 24 hours topical application Subject DHEP AssayDHEP Assay No. mg % Subject mg % 1 172.28 95.18 1left arm 169.54 93.67 2168.95 93.34 2left arm 169.61 93.71 3 173.20 95.69 3left arm 169.7293.77 4 176.13 97.31 4left arm 163.99 90.6 5 170.31 94.09 5left arm164.71 91.00 6 168.79 93.25 6left arm 169.48 93.64 7 177.23 97.92 7leftarm 167.79 92.70 8 174.63 96.48 8left arm 167.46 92.52 9 182.48 100.829left arm 169.22 93.49 10 185.88 102.70 10left arm 167.60 92.60 11173.04 95.60 11left arm 166.23 91.84 12 171.83 94.93 12left arm 167.4092.49 13 181.78 100.43 13right arm 165.46 91.41 14 174.84 96.60 14rightarm 165.27 91.31 15 170.17 94.02 15right arm 166.00 91.71 16 174.8296.59 16right arm 169.68 93.75 17 177.97 98.33 17right arm 171.52 94.7618 176.50 97.51 18right arm 168.74 93.23 19 178.64 98.70 19right arm170.01 93.93 20 187.61 103.65 20right arm 169.38 93.58 21 170.65 94.2821right arm 175.35 96.88 22 171.34 94.66 22right arm 171.10 94.53 23177.58 98.11 23right arm 170.76 94.34 24 166.88 92.20 24right arm 171.9494.99 Mean 175.15 96.77 Mean 168.66 93.19 RSD 5.35 RSD 2.60 CV % 3.06 CV% 1.60

The DHEP loss, after 24 hour of topical application, measure as % of thedeclared assay was about 3.6%.

Table 8 hereinbelow shows the heparin content value for 24Flector-Heparin, analyzed before topical treatment and after 24 hourstopical application in 24 volunteers.

TABLE 8 Time 0 After 24 hours topical application Subject Heparin AssayHeparin Assay No. IU % Subject IU % 1 5226 93.32 1right arm 5210 93.04 25010 89.46 2right arm 5311 94.84 3 5203 92.91 3right arm 5709 101.95 45917 105.66 4right arm 5782 103.25 5 5569 99.45 5right arm 6163 110.05 65246 93.68 6right arm 5822 103.96 7 5839 104.27 7right arm 61.02 108.968 5193 5193 8right arm 5408 96.57 9 5672 101.29 9right arm 5674 101.3210 5421 96.80 10right arm 5826 104.04 11 6105 109.02 11right arm 552298.61 12 5625 100.45 12right arm 5526 98.68 13 5297 94.59 13left arm5356 95.64 14 5163 92.20 14left arm 4819 86.05 15 5934 105.96 15left arm5420 96.79 16 5919 105.70 16left arm 5449 97.30 17 5801 103.59 17leftarm 5869 104.80 18 6052 108.07 18left arm 6251 111.63 19 5633 100.5919left arm 5517 98.52 20 5967 106.55 20left arm 6182 110.39 21 537295.93 21left arm 5699 101.77 22 5033 89.88 22left arm 5305 94.73 23 5712102.00 23left arm 5071 90.55 24 6125 109.38 24left arm 5920 105.71 Mean5585 99.73 Mean 5621 100.38 RSD 356.3 RSD 363.5 CV % 6.4 CV % 6.5

The heparin assay loss, after 24 hours of topical application, wasinconsistent. Form the above results it can be seen that heparin is notreleased from the plaster after topical application. Thus, after 24hours a loss of DHEP content was measured into test patches incomparison to the references patches demonstrating that DHEP wasabsorbed through the skin. After 24 hours topical application of patchesthe DHEP loss was, in facts, about 3.6%. On the contrary, the results onheparin assay loss, after 24 hours of topical application demonstratedthat heparin does not permeate in vitro. The same mechanism occurred invivo.

Example 4

Effects of a plaster containing diclofenac epolamine (DHEP) and heparinon the pain threshold in asymptomatic subjects (no spontaneous pain)with latent algogenic condition (subcutaneous and/or muscle hyperalgesiaand dystrophic changes) of the lower limb.

The study plan included three visits at the clinics, which took place atscreening/randomization (Visit T0/T1, day 1), after 3 days of medicatedplasters application (Visit T4, day 4) and after 7 days of medicatedplasters application (Visit T8, day 8, final visit).

Subjects of either sex, aged between 18 and 65 years, having nospontaneous pain since one month before the enrolment, with hyperalgesiaand trophic changes of deep somatic structures from latent algogenicconditions of the lower limbs.

A total number of 104 subjects were screened for enrolment in this studyand were randomized—equally distributed—to one of the four treatments:26 subjects (25% of total population) therefore constituted each group.

EFFICACY RESULTS: The four treatment groups were well matched fordemographic and other baseline characteristics, with gender distributionand thickness of subcutis being the only exceptions and were thereforeincluded as covariates in the statistical models. Noteworthy, the meanvalue of the baseline pain threshold to electrical stimulation of themuscle confirm the presence of a latent algogenic condition in theenrolled subjects, if compared to normal values.

The results of the primary variable ‘pain threshold to electricalstimulation of the muscle confirm the ability of the DHEP plaster toincrease the pain threshold of volunteers with latent algogeniccondition as compared to placebo (p=0.0299), in agreement with previousdata, and showed a significantly more marked increase in the DHEPHeparin group as compared to the other groups over the treatment periodboth in the ITT (Table 9) and PP population.

The difference in efficacy was statistically significant in favor of theDHEP Heparin group as compared to DHEP (p=0.0307), Heparin (p=0.002) andplacebo (p<0.0001) over the 7-day treatment period. Moreover, thedifference between Heparin and placebo was not significant (p=0.2568).Finally, for the pain threshold to electrical stimulation in cutis andsubcutis, the increase of pain thresholds was generally more marked forthe DHEP-containing medicated plasters as compared to those notcontaining DHEP, although the analyses did not reveal any statisticallysignificant difference between groups.

Table 9: Adjusted mean values for change from baseline of pain thresholdto electrical stimulation of muscle as compared to the mean valueswithout adjustment for the covariates.

TABLE 9 DHEP Muscle (mA) Heparin DHEP Heparin Placebo Day 4 (Mean ± SD)0.29 ± 0.38 0.15 ± 0.27 0.06 ± 0.33 −0.09 ± 0.43 Day 4 (Adjusted Mean0.27 0.13 0.03 −0.12 for Gender) Day 4 (Adjusted Mean 0.28 0.15 0.06−0.09 for Subcutis thickness) Day 8 (Mean ± SD) 0.40 ± 0.46 0.16 ± 0.380.06 ± 0.38 −0.02 ± 0.28 Day 8 (Adjusted Mean 0.38 0.14 0.03 −0.04 forGender) Day 8 (Adjusted Mean 0.40 0.17 0.06  0.00 for Subcutisthickness)

The comparison between groups on mean changes from baseline after 3 and7 days of treatment for both primary and secondary efficacy parameterswas performed by means of an ANCOVA model with baseline values as acovariate.

The compliance to study treatment was calculated and the comparisonsbetween groups were performed after 7 days of treatment by means of theChi² test. Adverse events were coded using MedDRA dictionary. The SystemOrgan Class (SOC) and Preferred Term (PT) were used for tabulation. Thedifference between treatment groups in the proportion of patients whoexperienced adverse events and serious adverse events was evaluatedusing Chi² test.

A descriptive analysis of vital signs (blood pressures and hearth rate)by-visit was performed. The results of the secondary variables ‘painthreshold to mechanical (pressure) stimulation in muscle’ and ‘thicknessof muscle at ultrasound examination’ did not show any statisticallysignificant differences between treatment groups during the studyperiod.

Although the ‘thickness of subcutis measurements at post-treatmentultrasound examination’ showed statistically significant differencesbetween heparin group and the other groups, the clinical relevance ofsuch a finding is not clear considering the presence of statisticallysignificant pre-treatment intergroup differences.

The results support a superior effect of the new DHEP Heparin plaster incomparison to the DHEP plaster in increasing the pain threshold toelectrical stimulation at muscle level on volunteers with latentalgogenic condition of the quadriceps muscle following a once-a-dayplaster application for 7 consecutive days. A 30% increase in painthreshold as compared to the pre-treatment value was reported in theDHEP Heparin treated subjects, while the increase was only 12% insubjects treated with DHEP plaster. Differently from a plastercontaining heparin only, both DHEP medicated plasters were found to besignificantly more effective than placebo.

We claim:
 1. Method of improving release of non-steroidalanti-inflammatory drugs (NSAIDs) from a bandage comprising: a) asubstrate layer; b) an adhesive layer in the form of a polymerichydrogel matrix comprising an effective amount of a pharmaceuticallyacceptable NSAID and heparin or a heparinoid, at least one thickeningagent, at least one wetting agent, at least one cross-linking agent; andc) a protective film which can be removed at the moment of use, whereinsaid hydrogel matrix further comprises a surfactant consisting of sodiumethylenediamine tetraacetate; said method comprising topically applyingsaid bandage to patients in need thereof; and assessing an improvedNSAID release compared NSAID release from bandages without heparin. 2.Method of increasing membrane permeation of non-steroidalanti-inflammatory drugs (NSAID) from a bandage comprising: a) asubstrate layer; b) an adhesive layer in the form of a polymerichydrogel matrix comprising an effective amount of a pharmaceuticallyacceptable NSAID and heparin or a heparinoid, at least one thickeningagent, at least one wetting agent, at least one cross-linking agent; andc) a protective film which can be removed at the moment of use, whereinsaid hydrogel matrix further comprises a surfactant consisting of sodiumethylenediamine tetraacetate; said method comprising: applying saidbandage to a membrane; and assessing an increased membrane permeation ofNSAID compared to membrane permeation obtainable from bandages withoutheparin.
 3. Method of reducing muscle hyperalgesia in subjects withoutspontaneous pain, said method comprising: assessing an initial painthreshold in said subjects; applying to said subjects a bandagecomprising: a) a substrate layer; b) an adhesive layer in the form of apolymeric hydrogel matrix comprising a pharmaceutically acceptable NSAIDand heparin or a heparinoid, at least one thickening agent, at least onewetting agent, at least one cross-linking agent; and c) a protectivefilm which can be removed at the moment of use, wherein said hydrogelmatrix further comprises a surfactant consisting of sodiumethylenediamine tetraacetate; reassessing the pain threshold; anddetermining a reduced muscle hyperalgesia in said subjects measured as adifference between the initial and the reassessed pain threshold.
 4. Themethod according to claim 1, wherein the NSAID is selected from thegroup consisting essentially of diclofenac, diflunisal, ibuprofen,ketoprofen, naproxen, acetysalicic acid, salsalate, celecoxib, etodolac,fenoprofen, flurbiprofen, indomethacin, ketorolac, meloxicam, mefenamicacid, nabumetone, oxaprozin, piroxicam, rofecoxib, sulindac, tolmetin,valdecoxib or pharmaceutically acceptable salts thereof.
 5. The methodaccording to claim 1, wherein the NSAID is diclofenac.
 6. The methodaccording to claim 4, wherein the diclofenac salt is in the form of asalt with an alicyclic amine of general formula

in which m is 0 or
 1. 7. The method according to claim 4, wherein thediclofenac salt is the salt of N-hydroxyethyl pyrrolidine.
 8. The methodaccording to claim 1, wherein the effective amount of NSAID is between0.1 and 5 wt % with respect to the hydrogel matrix.
 9. The methodaccording to claim 1, wherein the effective amount of NSAID is between0.3 and 3 wt % with respect to the hydrogel matrix.
 10. The methodaccording to claim 1 wherein the concentration of heparin or heparinoidis between 0.05 and 1 wt % with respect to the hydrogel matrix.